Intratumoural immune cell landscape in germinoma reveals multipotent lineages and exhibits prognostic significance.

AIMS: Alterations in microenvironments are a hallmark of cancer, and these alterations in germinomas are of particular significance. Germinoma, the most common subtype of central nervous system germ cell tumours, often exhibits massive immune cell infiltration intermingled with tumour cells. The role of these immune cells in germinoma, however, remains unknown. METHODS: We investigated the cellular constituents of immune microenvironments and their clinical impacts on prognosis in 100 germinoma cases. RESULTS: Patients with germinomas lower in tumour cell content (i.e. higher immune cell infiltration) had a significantly longer progression-free survival time than those with higher tumour cell contents (P = 0.03). Transcriptome analyses and RNA in-situ hybridization indicated that infiltrating immune cells comprised a wide variety of cell types, including lymphocytes and myelocyte-lineage cells. High expression of CD4 was significantly associated with good prognosis, whereas elevated nitric oxide synthase 2 was associated with poor prognosis. PD1 (PDCD1) was expressed by immune cells present in most germinomas (93.8%), and PD-L1 (CD274) expression was found in tumour cells in the majority of germinomas examined (73.5%). CONCLUSIONS: The collective data strongly suggest that infiltrating immune cells play an important role in predicting treatment response. Further investigation should lead to additional categorization of germinoma to safely reduce treatment intensity depending on tumour/immune cell balance and to develop possible future immunotherapies.

The pleiotrophin-ALK axis is required for tumorigenicity of glioblastoma stem cells.

Increasing evidence suggests that brain tumors arise from the transformation of neural stem/precursor/progenitor cells. Much current research on human brain tumors is focused on the stem-like properties of glioblastoma. Here we show that anaplastic lymphoma kinase (ALK) and its ligand pleiotrophin are required for the self-renewal and tumorigenicity of glioblastoma stem cells (GSCs). Furthermore, we demonstrate that pleiotrophin is transactivated directly by SOX2, a transcription factor essential for the maintenance of both neural stem cells and GSCs. We speculate that the pleiotrophin-ALK axis may be a promising target for the therapy of glioblastoma.

Extensive and preferential Fas/Fas ligand-dependent death of gammadelta T cells following infection with Listeria monocytogenes.

In the spleens of mice infected intraperitoneally with the bacterium Listeria monocytogenes, both alphabeta and gammadelta T cells became rapidly activated, followed by a massive apoptotic death response predominantly within the gammadelta population. The death response involved two major splenic gammadelta T-cell subsets and was Fas/Fas ligand (Fas-L)-dependent. Among T cells isolated from the Listeria-infected spleen, Fas-L was almost exclusively expressed in gammadelta T cells. gammadelta T cells coexpressed Fas and Fas-L, suggesting activation-induced suicide as a mechanism of their death. In vivo treatment with an antibody specific for CD3epsilon induced activation, preferential Fas-L expression and apoptosis of gammadelta T cells, resembling the response pattern in listeriosis, whereas antibodies specific for T-cell receptor-beta (TCR-beta) or TCR-delta did not, suggesting that the complete response seen in listeriosis requires both gammadelta TCR engagement and additional stimuli. L. monocytogenes causes early nonspecific, Fas-independent lymphocyte death in heavily infected tissues. In contrast, the death response described here is selective, Fas-dependent and triggered at low local levels of bacteria, suggesting that it is controlled by interactions with other infection-activated host cells, and perhaps part of a regulatory circuit specifically curtailing gammadelta T cells.

Mesh-and-glue technique to prevent leakage of cerebrospinal fluid after implantation of expanded polytetrafluoroethylene dura substitute--technical note.

Expanded polytetrafluoroethylene (ePTFE) can be used as a dura substitute but is associated with leakage of cerebrospinal fluid (CSF) through the suture line. Fibrin glue alone may not prevent this problem. This new method for sealing the suture line in ePTFE membrane uses an absorbable polyglycoic acid mesh soaked with fibrinogen fluid placed on the suture line. Thrombin fluid is then slowly applied to the wet mesh, forming a large fibrin membrane reinforced by the mesh over the suture line. Only one of 33 patients in whom this technique was used had CSF leakage, whereas 12 of 59 patients in whom a dural defect was closed with ePTFE alone showed postoperative subcutaneous CSF collection (p < 0.05). Our clinical experiences clearly show the efficacy of the mesh-and-glue technique to prevent CSF leakage after artificial dural substitution. Mesh and glue can provide an adequate repair for small dural defect. The mesh-and-glue technique may also be used for arachnoid sealing in spinal surgery.

Early preferential stimulation of gamma delta T cells by TNF-alpha.

Although recent findings indicate that gamma delta T cells influence both early innate and Ag-specific adaptive host responses, it has remained unclear what triggers gamma delta T cell reactivity. Investigating very early T cell activation in mouse and human models of bacterial infection, we measured CD69 expression as an indicator of early cellular activation. Both murine alpha beta and gamma delta T cells responded polyclonally to systemic bacterial infections, and to LPS. However, gamma delta T cells responded more strongly to the bacteria and to LPS. In vitro LPS-stimulated human T cells showed a similar differential response pattern. We identified TNF-alpha as mediator of the early differential T cell activation, and of differential proliferative responses. The stronger response of gamma delta T cells to TNF-alpha was correlated with higher inducible expression levels of TNF-Rp75. Among unstimulated splenocytes, more gamma delta T cells than alpha beta T cells expressed CD44 at high levels. The data suggest that TNF-Rp75 determines the differential T cell reactivity, and that most gamma delta T cells in the normal spleen are present in a presensitized state. As TNF-alpha stimulates activated T cells, it may early preferentially connect gamma delta T cell functions with those of cells that produce this cytokine, including activated innate effector cells and Ag-stimulated T lymphocytes.

Disruption of the cellular inflammatory response to Listeria monocytogenes infection in mice with disruptions in targeted genes.

The results of this study to dissect the nature of the acquired immune response to infection with Listeria monocytogenes in mice with targetted gene disruptions show that successful resolution of disease requires the essential presence of alphabeta T cells and the capacity to elaborate gamma interferon. In the absence of either of these entities, mice experience increasingly severe hepatitis and tissue necrosis and die within a few days. The data from this study support the hypothesis that the protective process is the efficient replacement of neutrophils in lesions by longer-lived mononuclear phagocytes; alphabeta-T-cell-knockout mice died from progressive infection before neutrophil replacement could occur, whereas in gammadelta-T-cell-knockout mice this replacement process in the liver has previously been shown to be much slower. In the present study we attribute this delay to reduced production of the macrophage-attracting chemokine MCP-1 in the gammadelta-T-cell-knockout animals. These data further support the hypothesis that gammadelta T cells are important in controlling the inflammatory process rather than being essential to the expression of protection.

Multilobular cavernous malformation: report of two cases.

The authors report two cases of cavernous malformation characterized by a multilobular appearance on magnetic resonance images. At surgery, the malformations consisted of several nests of angiomatous components that were separated by intervening brain tissue and connected with each other by tiny vessels. This basic configuration seems to explain the unexpected postoperative recurrence of cavernous malformations and/or rebleeding from the residual lesions.

Bacterial infection of the testis leading to autoaggressive immunity triggers apparently opposed responses of alpha beta and gamma delta T cells.

The mechanisms that lead to the breakdown of self-tolerance and testis-specific immune reactivity in the murine orchitis model are understood only in part. We investigated the histopathologic and immunologic consequences of a unilateral bacterial (Listeria monocytogenes) infection of the testis. Both infected and contralateral sides of this bilateral organ suffered severe inflammatory responses despite a conspicuous absence of bacteria in the contralateral tissue. Also, in both testicles, T cell populations increased, involving both alpha beta and gamma delta T cell subsets. Concomitant with the bilateral orchitis, testis-specific delayed type hypersensitivity and Ab responses developed. Ab depletion experiments indicated that in this orchitis model, as in others, alpha beta T cells are initiators of the autoaggressive reactivity. In contrast, Ab depletion of gamma delta T cells accelerated the inflammatory response in both testicles, suggesting a regulatory role for this type of T cells in both infection-induced and autoimmune orchitis.

Generation and characterization of a continuous line of CD8+ suppressively regulatory T lymphocytes which down-regulates experimental autoimmune orchitis (EAO) in mice.

We have previously shown that two injections with viable syngeneic testicular germ cells (TC) alone developed experimental autoimmune orchitis (EAO) in C3H/He mice, and that the induction of antigen-specific tolerance in this EAO model is associated with the generation of antigen-specific suppressively regulatory T (Ts) cells. For the elucidation of the nature of these Ts cells, a murine Ts cell line (designated Ts-A) was established. This line was generated from the spleen cells of C3H/He mice which had received three i.v. injections of a soluble (deaggregated) form of murine testicular antigen (mTA), followed by the repeated selection of these spleen lymphocytes in vitro by stimulation with mTA. Adoptive transfer of Ts-A cells into naive syngeneic mice immediately before the first TC injection was found to downgrade EAO in actively immunized recipients. The transferred Ts-A cells significantly inhibited the cellular immune response to TC in the recipients in an antigen-specific manner, but these cells had no inhibitory effect on the humoral immune response to TC. This line could also inhibit in vitro syngeneic TC-driven proliferation of orchitogenic lymphocytes. Surface phenotype of this line was CD8+, CD4-, Thy-1.2+, CD3+, and TCR alpha beta+. These findings may suggest an in vivo role for suppressively regulatory lymphocytes, capable of inhibiting helper T cells, in the regulation of EAO.

Genetic susceptibility to the induction of murine experimental autoimmune orchitis (EAO) without adjuvant. I. Comparison of pathology, delayed type hypersensitivity, and antibody.

In the present study, it was demonstrated that there were marked strain differences in susceptibility to the induction of our new murine model of experimental autoimmune orchitis (EAO; definite orchitis with hypospermatogenesis) induced by two or three sc injections with viable syngeneic testicular germ cells (TC) without any adjuvants. Among 12 inbred strains of mice examined, the A/J (H-2a), C3H/He (H-2k), and C3H/HeN (H-2k) strains were highly susceptible, whereas the C57BL/6N (H-2b), C57BL/10Sn (H-2b), BALB/cAnN (H-2d), AKR/N (H-2k), CBA/JN (H-2k), C3H/HeJ (H-2k), and MRL/lpr (H-2k) strains were low susceptible, and the DBA/2N (H-2d) as well as C3H/BiKi (H-2k) strains were resistant. In particular, mice of the H-2k haplotype demonstrated varying degrees of susceptibility, from highly to totally resistant, to the induction of EAO. Disease susceptibility to this type of EAO does not seem to be associated with a particular H-2 haplotype. All mice of the highly susceptible strains that received two injections of TC (TC x 2) developed a significant increase in both levels of delayed footpad reaction (DFR) to TC and anti-TC antibodies measured by ELISA. In the low susceptible and the resistant strains receiving TC x 2 or TC x 3, there was no correlation between the immune responses and the susceptibility to disease in these strains, with the exception of the BALB/cAnN mice receiving TC x 3. The low susceptible and the resistant mice that received TC x 2 were classified into four groups based on the DFR and antibody response: the C57BL/6N, BALB/cAnN, CBA/JN, and C3H/HeJ strains were both positive, and the C57BL/10Sn and AKR/N strains were both negative or very low; the DBA/2N and MRL/lpr strains showed negative DFR and positive antibody response, and the C3H/BiKi strain showed quite the opposite. Almost all mice of the 12 inbred strains that received TC x 3 showed positive antibody response, although its level varied. There seems to be no linkage between the cell-mediated and humoral immune responses and the H-2 locus in our new EAO model.

Suppression of efferent limb of testicular autoimmune response by a regulatory CD4+ T cell line in mice.

A murine T cell line (designated as C.Ts) as a mediator of suppression of experimental autoimmune orchitis (EAO) was established. The method of establishment of C.Ts cell line was preparing spleen cells from C3H/He mice hyperimmunized with testicular germ cells (TC) and the repeated selection of the lymphocytes in vitro by stimulation with mouse testicular antigens (mTA). The C.Ts cells were Thy1.2+, surface immunoglobulin-, CD3+, CD4+ and CD8-. The cells could suppress the induction of EAO when transferred into actively EAO-sensitized mice only at the pre-clinical stage of the disease (efferent limb of the autoimmune response). The transferred C.Ts cells significantly inhibited both cellular and humoral immune responses to TC in the recipients in an antigen-specific manner. The disease suppression by C.Ts cells was found to depend upon their cell number, and their suppressive activity was markedly augmented by in vitro stimulation with mTA.

Inhibition of a novel model of murine experimental autoimmune orchitis by intravenous administration with a soluble testicular antigen: participation of CD8+ regulatory T cells.

Recently, we established a novel murine model of experimental autoimmune orchitis (EAO) in C3H/He mice by means of two sc injections of 1 x 10(7) viable syngeneic testicular germ cells (TC) without the use of any adjuvants. Using this model, an effective and reproducible system of immunoregulation in EAO was developed. The induction of this EAO was suppressed by pretreatment with five iv injections of a soluble (deaggregated) form of murine testicular antigen (mTA). The antigen, mTA, was prepared by acid extraction and ammonium sulfate precipitation of defatted testes and epididymides. The development of EAO and relevant delayed-type hypersensitivity was suppressed in an antigen-specific fashion, but anti-TC antibody formation was not affected. A single dose of cyclophosphamide at 2 days after the tolerogenic regime abrogated the unresponsiveness to EAO. Three doses of recombinant interleukin 2 at every other day starting on the next day of the last pretreatment did not overcome the unresponsiveness to EAO. CD8+ T cells isolated from the spleen of deaggregated mTA-pretreated animals could adoptively transfer suppression against EAO into naive recipients, whereas CD4+ T cells failed to transfer the suppression.

New experimental model for adoptive transfer of murine autoimmune orchitis.

Previous studies demonstrated that experimental autoimmune orchitis (EAO) was produced in C3H/He mice with very high incidence by subcutaneous (s.c.) injections of viable syngeneic testicular germ cells (TC), without resorting to any adjuvants or immunopotentiators. Using this EAO model, a new and simple protocol was developed for adoptive transfer of EAO. Cell donors were C3H/He mice that received s.c. injections twice with TC alone. Spleen cells from the donors were stimulated in vitro with TC, propagated in interleukin-2 containing medium, then injected i.p. to naive recipient mice. This procedure induced severe orchitis and hypospermatogenesis with or without inflammation in epididymis and vas deferens in the recipients at high incidence. Elimination of all T cells or CD4+ T cells before the transfer produced no histopathological signs in the recipients whereas that of the CD8+ T cells or B cells had no inhibitory effect on the disease transfer, indicating that the effector cells are CD4+ T cells.

Establishment of a murine thymic epithelial cell line capable of inducing both thymic nurse cell formation and thymocyte apoptosis.

A thymic epithelial cell (TEC) line (B/c. TEC-L1) was established from a normal thymus of a 4-week BALB/c mouse. The B/c. TEC-L1 had an epithelial morphology showing a contact-inhibited cobblestone-like arrangement with occasional desmosome-like structures at the adjacent cellular membranes. B/c.TEC-L1 cells showed positive staining for desmosomal glycoprotein, cytokeratin, thymosin alpha 1 beta 3, and I-Ad, and MHC class I antigens. The doubling time was 24 hours, and the chromosome number ranged from 52 to 78 with the mode of 70. Coculture of B/c.TEC-L1 cells with syngeneic, peanut agglutinin-agglutinated (PNA+) thymocytes in suspension at 37 degree C was followed by the formation of TEC thymocyte rosettes, after which the reconstitution of thymic nurse cells ensued. At 4 degrees C, PNA+ thymocytes bound to the B/c.TEC-L1 cell but did not form thymic nurse cells. PNA- thymocytes, although to a lesser degree than PNA+ cells, bound to the TECs at 37 degrees C, but at 4 degrees C few cells bound to the TECs. Allogeneic thymocytes also bound to the TECs at 37 degrees C. When the PNA+ thymocytes were cultured on the B/c.TEC-L1 monolayer, the small ones chiefly adhered on the surface of the TECs, while underneath the TECs the relatively large thymocytes (including cells in mitosis) predominated. Although the PNA- thymocytes bound to the surface of the monolayer within a few hours after coculture, by 24 hours nearly all cells disappeared. It is presumed that the thymocytes creeping underneath the B/c.TEC-L1 monolayer and those enveloped within the thymic nurse cell reconstituted in the suspension culture; both may be placed in circumstances analogous to the thymic microenvironment, wherein immature thymocytes appear to contact TECs directly and to be exposed to higher concentrations of thymic hormones and other soluble factors. Additionally, cell death in the PNA+ thymocytes was also observed in the coculture with B/c.TEC-L1 cells. The PNA+ cells revealed the morphological changes termed "apoptosis" characterized by chromatin condensation and nuclear fragmentation.

Cerebrospinal irradiation of Burkitt's lymphoma. Failure in preventing central nervous system relapse.

Twenty-two patients with Burkitt's lymphoma in complete remission induced by either cyclophosphamide or a combination of cyclophosphamide, oncovin and methotrexate were randomized to receive or not to receive prophylactic cerebrospinal irradiation. Six of 11 irradiated patients relapsed with tumour of the central nervous system as compared to 4 of 11 controls. Relapse frequency appeared to be related to stage of disease on admission. It is concluded that irradiation does not prevent relapse.

Epstein-Barr virus genome studies in Burkitt's and non-Burkitt's lymphomas in Uganda.

Burkitt's lymphoma (BL) has been widely investigated and has attracted attention because of the possible etiologic role of the Epstein-Barr virus (EBV). To further determine the role of EBV in the causation of this tumor, we measured EBV-specific nuclear antigen (EBNA) and EBV DNA using immunofluorescence and nucleic acid hybridization techniques, respectively. Of 34 BL biopsies, 27 tissues (79%) were EBNA-positive, whereas none of the 25 non-BL biopsy tissues were EBNA-positive. Of 15 BL tumors tested, 14 (93%) were EBV DNA-positive with a mean of 39 (range, 8-86) EBV genome equivalents per cell. Each of the 15 non-BL biopsy specimens subjected to nucleic acid hybridization had less than two virus genome equivalents per cell, although all had serologic evidence of past EBV infection. The findings further supported the possible etiologic role of EBV in African BL and negated the passenger hypothesis. The EBV genome could, therefore, be used as a separating marker between African BL and non-BL lymphomas.

Treatment of Burkitt's lymphoma: randomized clinical trial of single-agent versus combination chemotherapy.

A randomized clinical trial designed to compare the effectiveness of cytoxan (CTX) alone versus a combination consisting of CTX, vincristine (Oncovin) and methotrexate (COM) in the treatment of Burkitt's lymphoma (BL) was carried out. Nineteen patients were selected at random to receive CTX alone while 21 received COM. The two treatment regimens were equally effective in inducing remissions, and complete response rates of 83.3% and 84.3% were observed for CTX- and COM-treated patients, respectively. The relapse frequencies were also equal but the pattern of relapse was clearly different. Seven out of 8 (87.5%) in the CTX group relapsed with systemic and central nervous system (CNS) tumor, while 8 out of 10 (80%) in the COM group relapsed with CNS disease only. This difference is highly significant p = 0.008. The remission durations and survival to date are the same.